The present invention relates to an immunoassay reagent utilizing an insoluble carrier and an immunoassay method, and more particularly to an immunoassay reagent and an immunoassay method capable of high-sensitive detection of a target substance in a subject.
In the field of clinical testing, diagnoses of diseases are carried out using biosamples (such as blood and urine). For these diagnoses, a variety of assays have been developed and utilized. Typical assays include biochemical assays such as utilizing an enzyme reaction, and immunoassays such as utilizing an antigen-antibody reaction. The recent demand to perform precise assaying of components present in biosamples has led to the wide-spread use of immunoassay methods utilizing highly-specific antigen-antibody reactions.
Examples of immunoassay methods include immuno-turbidimetry (TIA method), latex immunoassay (LIA method), enzyme immunoassay (EIA method) and radio-immunoassay (RIA method). Selection is made depending upon the particular purpose contemplated. That is, the TIA or LIA method may be employed when a biosample contains a target substance in a relatively large quantity. The TIA and LIA methods are generally employed to assay a substance, such as C-reactive protein (CRP), anti-streptolysin O antibody (ASO) or fibrin degradation product (FDP), particularly when it is contained in a biosample in concentrations of not below several ng/ml. On the other hand, the EIA or RIA method may be used when a biosample contains a target substance in a relatively small quantity. These TIA and LIA methods are generally employed in assaying substances, such as cancer markers represented by xcex1-fetoprotein (AFP) and hormones represented by insulin, particularly when they are contained in a biosample in concentrations of not above several ng/ml.
The recent trend of placing greater importance on assay of microscale substances in a biosample further increases frequencies in use of EIA and RIA methods. In contrast to the TIA and LIA methods which can enjoy shortend testing time and simplified operation and find applications to various types of autoanalyzers (hereinafter referred to as clinical chemistry autoanalyzers), the EIA and RIA methods suffer from the following dificiencies: they need prolonged reaction periods; they require complex operations; and they employ diverse enzymes and radioisotopes. Because of these dificiencies, the EIA and RIA methods are frequently limited to use with specific autoanlyzers (hereinafter referred to as specialized autoanalyzers). The RIA method further requires special facilities because of its utilization of radioisotopes.
A need has arisen for a technique which allows assaying of ultramicro-scale substances in a biosample to thereby enable early detection of cancers and early diagnosis of infection with AIDS virus. There are two groups of techniques which have been found to enable assaying of ultramicro-scale substances. One group of techniques is directed to increase precisions of conventional assay methods, including modifications and improvements of the LIA and EIA methods. Another group of techniques is directed to improve performances of conventional devices used for the LIA and EIA methods. Some of such techniques have been put into practice.
Examples of techniques contemplated to increase the precision of assay methods themselves include a technique which colors insoluble carriers for use in the LIA method (Japanese Patent Laying-Open No. Hei 1-214760) and a technique which utilizes fluorescent materials, instead of enzymes, for labeling antigens or antibodies for use in the EIA method (Japanese Patent Laying-Open No. Hei 5-34346). Also, examples of techniques contemplated to improve performances of deviced include a technique proposed in Japanese Patent Laying-Open No. Hei 3-167475.
However, neither of these techniques are applicable to clinical chemistry autoanalyzers and the problem of requiring specialized autoanalyzers remains unsolved. The need of such specialized autoanalyzers arises because the reaction time, procedure and type of enzyme or radioisotope for use in micro-scale assay methods, as represented by the EIA and RIA methods, are varied depending upon the particular method used, as stated earlier. Other major reason is based on the fact that the micro-scale assays as currently developed or heretofore marketed always require an operation called B/F separation (B is a bound component via an immune reaction and F is a free component). This makes them unapplicable to clinical chemistry autoanalyzers incapable of B/F separation and necessitates specialized autoanalyzers capable of B/F separation.
Assay methods which do not require B/F separation have been recently proposed and developed, as seen in Japanese Patent Laying-Open Nos. Hei 5-249112 and Hei 7-179495. Due to the insufficient sensitivity and extended determination period, they in some cases require specialized autoanalyzers and in other cases find a limited application to clinical chemistry autoanalyzers, which have been problems.
In order to perform ultramicro-scale analysis in the scene of actual clinical examinations, it is required to purchase an expensive special autoanalyzer and secure a place for its installation. There accordingly remains a strong demand for a method which can assay ultramicro-scale substances with the use of clinical chemistry autoanalyzers.
As stated above, the methods currently developed or marketed for assaying ultramicro-scale substances require the B/F separation, as against such a strong demand from users. This presents a major problem, i.e., their practices are limited on special autoanalyzers.
In the scene of clinical examinations, it is frequent to test several items using a single biosample. In such an instance, the single biosample is repeatedly subjected to different assay methods. This not only prolongs the time necessary to complete the testing, but also increases a chance for a tester to contact the biosample and thereby increases a risk of infection, which have been problems.
It is an object of the present invention to provide an immunoassay reagent and an immunoassay method which can solve the above-described problems, which can assay an ultramicro-scale substance in a sample, such as an antigen or antibody, with a high level of sensitivity, and which can either eliminate the need to carry out the B/F separation or simplify the B/F separation in assaying ultramicro-scale substances.
A first invention of the present application is an immunoassay reagent for quantitatively determining a target material, i.e., an antigen or antibody in a sample, which is characterized as containing (a) an insoluble carrier for carrying an enzyme and an antibody or antigen corresponding to the aforementioned antigen or antibody, the aforementioned insoluble carrier comprising at least one selected from the group consisting of an organic polymer powder particle, microorganism, blood cell and cell membrane fragment, (b) an enzyme inhibitor for inhibiting the activity of the aforementioned enzyme and (c) a substrate with which the aforementioned enzyme reacts.
In a particular aspect of the first invention, there are provided a first reagent containing the aforementioned insoluble carrier and a second reagent containing the aforementioned enzyme inhibitor and substrate.
A second invention of the present application is an immunoassay reagent for quantitively determining a target material, i.e., an antigen or antibody in a sample, which is characterized as containing (a) an insoluble carrier for carrying an enzyme inhibitor and an antibody or antigen corresponding to the aforementioned antigen or antibody, the aforementioned insoluble carrier comprising at least one selected from the group consisting of an organic polymer powder particle, microorganism, blood cell and cell membrane fragment, (b) an enzyme whose activity is inhibited by the aforementioned enzyme inhibitor and (c) a substrate with which the aforementioned enzyme reacts.
In a particular aspect of the immunoassay reagent in accordance with the second invention, there are provided a first reagent containing the aforementioned insoluble carrier, a second reagent containing the aforementioned enzyme and a third reagent containing the aforementioned substrate.
For the immunoassay reagents in accordance with the first and second inventions, a magnetic or magnetizable material is preferably incorporated in the insoluble carrier.
Also in the immunoassay reagents in accordance with the first and second inventions, the aforementioned antibody or antigen, enzyme inhibitor and enzyme can be used in several combinations whereby several types of antigens or antibodies can be quantitatively determined.
A third invention of the present application is an immunoassay reagent for quantitively determining a target material, i.e., an antigen or antibody in a sample, which is characterized as containing (a) an antibody or antigen corresponding to the aforementioned antigen or antibody and chemically coupled to an enzyme inhibitor, (b) an enzyme whose activity is inhibited by the aforementioned enzyme inhibitor and (c) a substrate with which the aforementioned enzyme reacts.
In a particular aspect of the third invention, there are provided a first reagent containing the aforementioned antibody or antigen chemically coupled to the enzyme inhibitor, a second reagent containing the aforementioned enzyme and a third reagent containing the aforementioned substrate.
In a particular aspect of the immunoassay reagent in accordance with the first, second or third inventions, an antibody against the enzyme is used as the enzyme inhibitor. In a more particular aspect, a monoclonal antibody is used.
A fourth invention of the present application is an immunoassay method utilizing the immunoassay reagent in accordance with the first, second or third invention. In this immunoassay method, a sample containing an antigen or antibody as a target material is mixed with the immunoassay reagent in accordance with the first, second or third invention, so that an agglutination reaction in the form of a antigen-antibody reaction and an enzyme reaction are caused to occur. The antigen or antibody can be quantitated by measuring the degrees of such reactions caused.
The first invention provides an immunoassay reagent which contains (a) an insoluble carrier that carries an enzyme and an antibody or antigen, (b) an enzyme inhibitor and (c) a substrate. Mixing of these components, prior to use, may cause an enzyme-substrate reaction to proceed or allow the enzyme inhibitor to deactivate the enzyme. Thus, the immunoassay reagent in its general form comprises two separate reagents; a first reagent containing the insoluble carrier (a) and a second reagent containing the enzyme inhibitor and substrate.
The below-described first and second reactions are caused to proceed when the first reagent containing the insoluble carrier that carries an enzyme and an antibody or antigen corresponding to the antigen or antibody as a target material in a sample, together with the second reagent containing a substance for inhibiting the activity of the enzyme (hereinafter referred to as an enzyme inhibitor) and the substrate with which the enzyme reacts, are mixed with a biosample containing the above-specified target material. The first reaction is an antigen-antibody reaction of the antigen or antibody present in the biosample with the corresponding antibody or antigen carried by the insoluble carrier. The first reaction is similar in principle to the LIA method and results in the agglutination of the insoluble carriers, whereby the mixture is increased in turbidity to change its light absorbence. On the other hand, the second reaction is a reaction between the enzyme and substrate and is similar in principle to the EIA. The absorbence of mixture is varied as the substrate undergoes a change.
Since the first and second reactions are caused to occur almost concurrently but independently, the degree of change in absorbence of the mixture is increased compared to the LIA method. This enables assaying of micro-scale substances present in a biosample. However, while the first reaction undergoes a change in absorbence with the amount of antigen or antibody present in the biosample, the second reaction is independent of the amount of antigen or antibody in the biosample because it is an enzyme-substrate reaction.
The inventors of the present application have found from their intensive researches that the inclusion of enzyme inhibitor in a reaction system induces the second reaction to depend upon the the amount of antigen or antibody present in the biosample. The enzyme inhibitor, when coupled to an enzyme, renders the enzyme inactive or less active. In the absence of the antigen or antibody in the biosample, the aggregation of insoluble carriers, via the first reaction, does not take place. The enzyme inhibitor is then allowed to bind to the enzyme on the insoluble carrier to render it inactive, so that the absorbence becomes unaffected by the second reaction. In contrast, in the presence of the antigen or antibody in the biosample, the first reaction results in the aggregation of insoluble carriers. In such an instance, a steric hindrance of the resulting aggregates reduces the occurrence of the enzyme inhibitor to bind to the enzyme on the insoluble carriers that participate in the aggregation. The enzyme is thus prevented from being deactivated and allowed to react with the substrate, thereby causing the change in absorbence.
In the fashion as stated above, the inclusion of the enzyme inhibitor in the reaction system renders the second reaction dependent upon the amount of antigen or antibody present in the biosample. Because the first and second reactions are both made dependent upon the amount of antigen or antibody present in the biosample, the immunoassay method in accordance with the first invention shows the increased sensitivity relative to the LIA method. Unlike the EIA method, it does not require the B/F separation or may be accompanied by the simplified B/F separation.
In the second invention, an immunoassay reagent is used which contains (a) an insoluble carrier that carries an antigen or antibody and an enzyme inhibitor, (b) an enzyme and (c) a substrate. Like the first invention, pre-mixing of these components may cause an enzyme-substrate reaction to proceed or the enzyme to deactivate.
Hence, the immunoassay reagent of the second invention in its general form comprises a first reagent containing the insoluble carrier, a second reagent containing the enzyme and a third reagent containing the substrate. In such a general form, if the first reagent is mixed with a biosample containing the above-specified target material, an antigen-antibody reaction (first reaction) is caused to occur between the antigen or antibody present in the biosample and the antibody or antigen carried by the insoluble carrier, resulting in aggregation of the insoluble carriers. When the second reagent is subsequently added, a reaction (second reaction) is caused to occur between the enzyme in the second reagent and the enzyme inhibitor supported by the insoluble carrier. This second reaction is dependent upon the amount of the antigen or antibody in the biosample. The enzyme, when reacted with the enzyme inhibitor, lose or reduce its activity. In the absence of the antigen or antibody in the biosample, the aggregation of the insoluble carriers via the first reaction does not occur. The enzyme inhibitor carried on the insoluble carrier is then allowed to attack the enzyme to render it inactive. Thus, the subsequent addition of the third reagent does not induce the absorbence change via the second reaction.
By contrast, in the presence of the antigen or antibody in the biosample, the insoluble carriers are caused to aggregate via the first reaction to the extent that depends upon the content of the antigen or antibody. In such a case, a steric hindrance of the resulting aggregates reduces the occurrence of the reaction between the enzyme and the enzyme inhibitor on the insoluble carrier. The enzyme is accordingly prevented from being deactivated. Therefore, when the third reagent is subsequently added, the enzyme is allowed to react with the substrate, whereby the absorbence is changed.
In the manner as stated above, the configuration of the insoluble carrier to support the enzyme inhibitor is also effective to render the second reaction dependent upon the amount of the antigen or antibody present in the biosample. Also in accordance with the immunoassay reagent and method of the second invention, the first and second reactions are both rendered dependent upon the amount of antigen or antibody present in the biosample.
Like the first invention, the immunoassay reagent and method can be obtained which have the increased sensitivity relative to the LIA method and which, unlike the EIA method, do not require the B/F separation or may be accompanied by the simplified B/F separation.
The immunoassay reagent in accordance with the first or second invention may further include a magnetic material or magnetizable material incorporated in the insoluble carrier. In the case where such a magnetic or magnetizable material is incorporated in the insoluble carrier, the insoluble carrier can be separated from the solution by operating an external magnet or magnetizable substance, after completion of all the reactions, so as to magnetically attract the insoluble carrier from a bottom portion of a reactor. This allows measurement in color of the solution only, without being affected by the increase in turbidity of the insoluble carrier.
Also for the immunoassay reagent in accordance with the first or second invention, the aforementioned antibody or antigen, enzyme inhibitor and enzyme may be used in several combinations whereby several types of antigens or antibodies can be quantitatively determined.
Also in the case where a magnetic or magnetizable material is included in the insoluble carrier and where the antibody or antigen, enzyme and enzyme inhibitor are used in several different combinations, the colors of individual enzymes only can be measured without being affected by a turbidity increase resulting from aggregation of the insoluble carriers. This allows simultaneous measurement of two or more types of antigens or antibodies.
The immunoassay reagent of the first invention does not necessarily comprise the aforementioned first and second reagents. Likewise, the immunoassay reagent of the second invention does not necessarily comprise the aforementioned first, second and third reagents. For the immunoassay reagent of the first invention, the aforementioned insoluble carrier (a), enzyme inhibitor (b) and substrate (c) may be mixed simultaneously and then added to a sample to be tested without delay, for example. Likewise, all the ingredients of the immunoassay reagent according to the second invention may be mixed simultaneously and then added to a sample to be tested without delay. As such, if conditions are properly set, the immunoassay reagent according to the first or second invention does not necessarily take the form of consisting of the separately-prepared first and second reagents or the separately-prepared first, second and third reagents.
In the immunoassay reagent according to the third invention, an enzyme inhibitor is chemically bound to an antibody or antigen. The first reaction is initially caused to occur. That is, when a biosample is mixed with the antibody or antigen (hereinafter referred to as a conjugate) corresponding to a target antigen or antibody present in the sample and chemically bound to the enzyme inhibitor, an agglutination reaction is caused to occur between the antibody or antigen and the antigen or antibody present in the biosample. This agglutination reaction results in creating a steric hindrance or changing a conformation of the enzyme inhibitor in the conjugate, whereby the enzyme inhibitor is rendered inactive and accordingly the enzyme inhibiting action is weakened. That is, the enzyme inhibiting action is weakened depending upon the level of agglutination via the antigen-antibody reaction.
As the second reaction is then caused to proceed, the enzyme inhibitor, according to its activity, restricts the action of enzyme so that the enzyme present in a system is deactivated to the extent that depends upon the activity of the enzyme inhibitor.
When the enzyme is finally allowed to react with the substrate, color emission occurs as the third reaction. By measuring the degree of such color emission, the degree of activity loss of the enzyme can be detected. That is, the degree of agglutination can be detected by finally measuring the enzyme activity from the substrate.
Summarizing the precedings, the occurrencce of an agglutination reaction, i.e., the antigen-antibody reaction between the target material and the conjugate, weakens the enzyme inhibiting action of the enzyme inhibitor that exists in the conjugate. Thereafter, the enzyme is deactivated by the action of enzyme inhibitor to the extent that depends upon the degree of agglutination. By measuring the activity of enzyme with the addition of the substrate, the target material in the system- can be assayed.
As explained above, in accordance with the immunoassay reagent and method of the present invention, the first, second and third reactions are all rendered dependent upon the amount of antigen or antibody present in a biosample. Therefore, they are novel immunoassay reagent and method which exhibit the increased sensitivity relative to the LIA method and which, unlike the EIA method, do not require the B/F separation.
In the preceding descrioptions, the first, second and third reactions are separately explained in three stages for better understanding. However, the reagent for use in the actual measurement is not necessarily divided into three types. If proper conditions are selected, the reagent may be divided into one or two types, or alternatively, into three or more types.
The target materials which can be assayed in the first, second or third invention may be antigens or antibodies contained in a biosample, examples of which include, but not limited to, hepatitis (B, C)-derived antigens or antibodies; HIV antigens or antibodies; syphilis-derived antigens or antibodies; cancer markers represented by xcex1-fetoprotein; hormones represented by insulin; autacoids and the like.
Examples of insoluble carriers for use in the first or second invention include powder-form organic polymers, microorganisms, blood cells, cell membrane fragments and the like. Examples of powder-form organic polymers, include powder-form natural polymers such as insoluble agarose, cellulose and insoluble dextran; powder-form synthetic polymers such as polystyrene, styrene-styrene sulfonic acid (sulfonate) copolymer, styrene-methacrylic acid copolymer, acrylonitrile-butadiene-styrene copolymer, vinyl chloride-acrylate copolymer, vinyl acetate-acrylate copolymer and the like. Particularly preferred is a latex in which synthetic polymer particles are uniformly suspended. While varied in type depending upon the particular end purpose and use contemplated, the insoluble carrier is generally produced by chemical synthesis or commercially avialable. Also suitable is the insoluble carrier having a sulfonic- or carboxyl-introduced surface. The latex, if used, preferably contains particles having sizes in the range of 0.05-1.5 xcexcm, more preferably in the range of 0.1-0.6 xcexcm.
In the first or second invention, a magnetic or magnetizable material may be incorporated in the insoluble carrier. Illustrative of the magnetic material is ferrite and illustrative of the magnetizable materials is iron oxide. A specific example of the magnetizable material-containing insoluble carrier is a product manufactured by Belytus Co., Ltd. and designated in trade as DYNABEADS.
The enzyme for use in the immunoassay reagent according to the first, second or third inventions is not particularly specified, so long as its reaction with a substrate results in the change in absorbency. Examples of enzymes include, but not limited to, peroxidase, alkaline phosphatase, xcex2-galactosidase and the like. Enzymes obtained either from natural sources or by a gene engineering technique are useful. In general, those obtained from natrual sources may be used conveniently.
The enzyme when in use for measurement may be diluted with a suitable buffer. Examples of buffers include, but not limited to, phosphate, tris, glycine and Good""s buffers. The type of the buffer may be suitably chosen depending upon the properties of the enzyme and substrate used. The enzyme when in use may preferably be adjusted to a concentration range of 0.001-10 IU/mL. However, such a range may be varied depending upon the particular type of the enzyme used.
The substrate for use in the immunoassay reagent according to the first, second or third inventions is a substance which provides an absorbency change when reacted with the enzyme used. In an exemplary case where peroxidase is used as the enzyme, a suitable substrate may be an aqueous hydrogen peroxide to which N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, o-phenylenediamine or pyrogallol is added. In another exemplary case where alkaline phosphatase is used as the enzyme, a suitable substrate may be p-nitrophenyl phosphate. In still another exemplary case where xcex2-galactosidase is used as the enzyme, a suitable substrate is o-nitrophenyl-xcex2-D-galactopyranoside. However, the substrate is not particularly specified in type and may be suitably chosen depending upon the particular purpose and use contemplated.
In general, the aforementioned substrates are either manufactured by chemical synthesis or available from the market. The substrate when in use for measurement is dissolved in or diluted with a suitable buffer. Examples of buffers include, but not limited to, phosphate, tris, glycine and Good""s buffers. The type of the buffer used may be suitably chosen depending upon the properties of the enzyme and substrate used. The substrate when in use may preferably be adjusted to-a concentration range of 0.1-1,000 mM. Such a range may however be varied depending upon the particular type of the substrate used.
The enzyme inhibitor for use in the immunoassay reagent according to the first, second or third inventions is not particularly specified, so long as it is able to couple to the enzyme used so that the enzyme is rendered inactive. Examples of useful enzyme inhibitors include peptides, antibodies, fluorine compounds, sulfur compounds and the like. The enzyme inhibitor may be suitably chosen depending upon the particular type of the enzyme used.
Where an antibody to the enzyme (hereinafter referred to as the anti-enzyme antibody) is used as the enzyme inhibitor, such an antibody may be either polyclonal or monoclonal in type and can be manufactured by any known technique. The anti-enzyme antibody, if polyclonal, can be immune produced from the enzyme introduced into an animal such as a rabit, goat or sheep. In the similar manner, the monoclonal antibody can also be produced by using any known technique.
The antibody such obtained may be suitably purified as by a known chromatography. If circumstances permit, it may be used without being subjected to special purification. The anti-enzyme antibody when in use for measurement is diluted as with a suitable buffer. Examples of buffers include, but not limited to, phosphate, tris, glycine and Good""s buffers. The buffer may be suitably chosen depending upon the properties of the enzyme and substrate used.
The enzyme inhibitor when in use may preferably be adjusted to a concentration range of 0.01-10 mg/mL. Such a range may however be varied depending upon the particular type of the enzyme inhibitor used.
The following describes a procedure which can be utilized to prepare the insoluble carrier (a) for use in the first invention, which carries an enzyme and an antibody or antigen corresponding to an antigen or antibody present in a biosample.
While varied depending upon the particular types of the enzyme and antibody or antigen used, the following technique is generally utilized to bind the antibody or antigen and enzyme to the insoluble carrier. A solution containing the antibody or antigen and a solution containing the enzyme are added simultaneously or sequentially to a suspension of insoluble carriers. The subsequent stirring causes the antibody or antigen and enzyme to bind to the insoluble carriers by physical adsorption.
In the case of insoluble carriers having a sulfonic- or carboxyl-introduced surface, the antibody or antigen and enzyme can be bound thereto by the addition of a suitable crosslinking agent. In this case, the antibody or antigen and enzyme must be chemically modified so that crosslinking can be achieved by a crosslinking agent. Considering the properties and structures of the antibody or antigen and enzyme used, a technique may be suitably selected whereby they are caused to physically adsorb or bind to the insoluble carriers with the aid of crosslinking agent.
The above-described binding reaction is preferably carried out in the pH range of 3-10 at a temperature of 2-50xc2x0 C. If the pH falls outside the specified range, a problem may arise, e.g., the antibody or antigen may undergo a change in property as it is a protein. If the reaction temperature falls below 2xc2x0 C., the reaction rate may become slow to result in the difficulty to obtain a product having a desired level of sensitivity. If the reaction temperature goes beyond 50xc2x0 C., a problem such as a property change of the antibody or antigen may arise.
In the preparation of the insoluble carrier (a), the enzyme bound thereto in the first invention is replaced by the enzyme inhibitor in the second invention.
The immunoassay reagent in accordance with the third invention includes the enzyme inhibitor chemically coupled to the antibody or antigen. The following procedure can be utilized to prepare such an enzyme inhibitor chemically coupled to the antibody or antigen.
While varied depending upon the types of the antibody or antigen and enzyme inhibitor, an optimum process whereby the enzyme inhibitor is chemically coupled to the antibody or antigen corresponding to a target antigen or antibody may be suitably chosen from conventionally-known processes. Examples of applicable processes include a mixed acid anhydride process wherein a carboxyl group is caused to react with ethyl chlorocarbonate or butyl chlorocarbonate to thereby derive active mixed acid anhydride which is subsequently reacted with an amino group of the other to form an amide bond; an active ester process wherein a carbodiimide-based condensing agent is used to convert a carboxyl group to an active ester form which is subsequently caused to react with an amino group of the other; a process utilizing glutaric aldehyde; a process utilizing periodic acid and the like. In the case where a polyclonal or monoclonal antibody is used as the enzyme inhibitor, a binding ratio of the antibody or antigen used to the enzyme inhibitor, (antibody or antigen):(enzyme inhibitor)=preferably 20:1-1:1, more preferably 10:1-1:1, still more preferably 5:1-1:1.
In accordance with the immunoassay method of the present invention, the immunoassay reagent according to the first, second or third invention is mixed with a test sample containing an antigen or antibody to cause an agglutination reaction as one type of an antigen-antibody reaction and an enzyme reaction. The antigen or antibody can be quantitated by measuring the degrees of these reactions.
While not limiting, a method which involves detecting optical properties of reaction products is generally utilized to measure such degrees of reactions. The most popular optical detection method involves detecting a change in color hue in response to light absorption. Other useful methods utilize fluorescence, chemiluminescence and bioluminescence. The optical measurement method is not particularly specified and may be suitably chosen depending upon the purpose and use contemplated. Examples include wavelength measurement, time-resolved fluorescence method and the like.
In the wavelength measurement, a useful wavelength typically falls within the approximate range of 250-1,000 nm. In this measurement method, an antigen-antibody reaction and an enzyme reaction are carried out under ordinary conditions. Various buffers can be used as a reaction medium. Any type of buffer can be used, so long as it has such ionic strength and pH that neither deactivate an antigen or antibody present in a biosample nor inhibit the antigen-antibody reaction and enzyme reaction. Examples of useful buffers include phosphate, tris and glycine buffers. A reaction temperature is preferably in the range of 10-50xc2x0 C., more preferably in the range of 20-40xc2x0 C.
FIG. 1 is a calibration curve obtained from Example 1 wherein an ordinate axis indicates a variation of absorbency at 600 nm and an abscissa axis indicates a titer (I.U./ml) of an HBs antigen in serum.
FIG. 2 is a calibration curve obtained from Example 2 wherein an ordinate axis indicates a variation of absorbency at 600 or 420 nm and an abscissa axis indicates an HBs antigen titer (I.U./ml) or a CRP concentration (mg/dl) in serum.